Determination of potassium ions in fluids

ABSTRACT

A process and a reagent for the determination of ions in fluids, wherein the influence of these ions on the activity of an enzyme is measured. The ions for example are sodium, potassium, calcium, magnesium, manganese, lithium, lead, zinc, copper, iron or other heavy metals or non-metallic ions comprising chloride, bicarbonate, protons, ammonium and substances that give rise to ammonium. The enzymes which are used may be a transferase, a hydrolase, an oxidoreductase or a lyase. An essential feature is a method to exclude interferences by ions by masking the interfering ions with a binding agent.

This application is a continuation of application Ser. No. 07/958,534,filed Oct. 8, 1992, now abandoned, which is a continuation applicationof Ser. No. 07/804,820 filed Nov. 27, 1991 now abandoned which is adivisional application of Ser. No. 07/696,326 filed Apr. 30, 1991, nowabandoned, which is a continuation application of Ser. No. 07/302,799filed Jan. 19, 1989, now abandoned.

This invention is concerned with methods and reagents for thedetermination of ions, hereinafter also called analytes, in biologicaland non-biological fluids.

The invention is based on the ability of many analytes to stimulate orinhibit the activity of a sensitive enzyme. The analytes may be cationsor anions, metallic or non-metallic, simple or compound. In practice itis frequently found that the analyte is present in the sample at aconcentration that lies outside the range of sensitivity of the relevantanalytical indicator enzyme, or that interference is caused by thepresence of other ions to which the enzyme is also sensitive. Thisinvention addresses and solves these problems in diverse ways.

In the practice of Clinical Biochemistry the measurement of serumelectrolytes are the most common analytical tests performed withinhospitals. These measurements are requested not only for routineinvestigations but frequently for emergency and life-threateningsituations where speed of analysis is essential. Since a major source ofdelay in hospitals is the transport of specimens from the wards to thediagnostic laboratories, a method that is easily performed near thebed-side would be of particular value in emergency situations.

A common method of analysing potassium and sodium in clinicalbiochemistry practice is flame photometry. This process depends on theprinciple that certain atoms when energized by heat become excited andemit light of a characteristic wavelength when returning to groundstate. The intensity of the characteristic wavelength of radiant energyproduced by the atoms in the flame is directly proportional to thenumber of atoms excited in the flame, which is directly proportional tothe concentration of the substance of interest in the sample. Theapparatus required is complex and relatively expensive and requires theuse of combustible gases.

An alternative method especially for sodium, potassium and chloridemakes use of ion-selective electrodes. Ideally, each electrode wouldpossess a unique ion-selective property that would allow it to respondto only one ion. In practice this is not the case and interfering ionsexist for all ion-selective electrodes. Moreover, ion-specificelectrodes are not absolutely specific although generally correctionsare possible. The electrodes measure the potential developed in thepresence of the specific ion. The instrumention is relatively expensive.Neither method can be performed spectrophotometrically and the clinicalneed for ion measurement therefore results in a substantial increase inthe complexity of commercially available clinical analysers, most ofwhich are designed primarily for spectrophotometric assays. Both methodsrequire a considerable degree of skill and knowledge for theirsuccessful implementation.

Similarly, the routine determination of chloride by coulometric methodsrequires special instrumentation. The endpoint of this titrationprocedure is detected by an increase in electrical flux completion offormation of insoluble silver chloride product. Alternatively,potentiometric determinations may be used which are also very timeconsuming and involve additional instrumentation.

For chloride, in addition, there are a number of photometric andtitrimetric methods, which e.g. include:

titrimetric determination of free Hg²⁺ ions via diphenylcarbazonecomplex

colorimetric determination of the rhodanide complex of iron, which isformed after dissociation of the mercury complexes upon precipitation ofHgCl₂ (Skeggs, Clin. Chem. 10, 1964, 918f.; Schmidt, Zentralblatt Pharm.124 (9), 1985, 527f)

colorimetric determination of chloranilic acid from the respectivemercury salt (Renschler)

determination of the coloured Cu²⁺ complex of diethyldithiocarbaminicacid from the colourless mercury salt (German Offenlegungsschrift2137146)

the rather common TPTZ-method (tripyridile-s-triazine) (R: Fried,Zeitschr. Klin. Chem., Klin. Bioch. 10, 1972, 280f; DOS 215 3387) whichis similarily based on the formation of a coloured metal complex upondissociation of a mercury complex.

A major drawback of these methods is the use of solutions containinghighly toxic substances. Some of the methods are complicated andimprecise (e.g. the titration method). Many of the reagents are unstableand calibration curves are non-linear (e.g. the rhodanide method). Someof these methods in addition need a pretreatment in order to eliminateinterferences by the protein content of the sample.

An improved TPTZ-method is described in No. 83/002670, the use of toxicmercury compounds however is still a disadvantage. The only colorimetricmethod without use of mercury ions is the determination of hexachlorocomplexes of Fe(III) in a perchloric acid solution (F. Hoppe., Ther.Ggw. 110(4), 1971, 554f.; H. Mahner, Zeitschr. Klin. Chem., klin.Biochem. 11(11), 1973, 451f.; W. T. Law, Clin. Chem. 26 (13) 1980,1874f.; U.S. Pat. No. 4,278,440). A considerable limitation of thismethod is the use of strongly acid reagents which are corrosive andtherefore not compatible with mechanical pipetting systems. A furtherdisadvantage is the interference by bilirubin in the samples.

Calcium is a further example of an electrolyte which is routinelydetermined in the clinical laboratory. The concentration of this metalion in body fluids is regulated within a narrow range. Pathologicallyhigh or low concentrations can lead to life threatening disorders suchas renal insufficiency, pancreatitis, tetany and congestive heartfailure.

One of the earliest methods for the determination of calcium was thatdescribed by Tisdall (J. Biol. Chem. 63,461-465, 1925) in which calciumis precipitated by oxalic acid which is in turn estimatedcolorimetrically. The method involves a centrifugation step and istherefore very time consuming; it is not specific for calcium anddepends on a careful handling of samples. The method has been succeededin many laboratories by titrimetric and direct colorimetric procedures.The former also has the drawback of a complicated and cumbersomeprocedure and requires large sample volumes. In the latter procedurecalcium affects the colour of a dye, for example orthocresolphthaleincomplexone, which can be measured in a photometer. Due to the simplicityof the method it lends itself to automation in the clinical laboratory.The method, however, involves the use of aggressive, highly alkalinesolutions and toxic substances. It is particularly prone to interferenceby a number of serum components such as lipids, proteins, phosphate andbilirubin and as a result does not agree well with atomic absorption andflame photometric reference methods. A further disadvantage of thecolorimetric procedure is that the calibration curves are non-linear andthe colour is greatly dependent on temperature. In W. H. Outlaw and O.H. Lowry, Analytical Biochemistry 92,370-374 (1979) an enzyme-mediatedassay for measuring potassium ions in tissues is described. The methodemploys pyruvate kinase, from rabbit muscle, which is activated bypotassium ions and sodium ions, the former being about one forty-foldmore effective. Because of this non-specificity the method may besuitable for plant material in which potassium ions are the predominantcations, but it is unsuitable for measurements in body liquids likeserum which contains a thirty-fold excess of sodium ions. Thereforesodium ions cause unacceptable interference when using the enzymaticphotometric technique as described by Lowry et al. to measure potassiumin plasma or serum. A further problem is that ammonium ions give asimilar activation to potassium ions. The above mentioned publicationdoes not address or solve these critical problems in regard to theanalysis of potassium ions in biological fluids such as serum, nor doesit propose any method for the determination of sodium ions.

Therefore, it is an object of the present invention to provide a processand a reagent by which the above mentioned problems are avoided. Theinvention solves the problems by a process for the determination of ions(analytes) in fluids wherein the influence of these ions on the activityof an enzyme is measured.

A key feature of this invention is the use of selective binding agentsto bring the free concentration of the analyte within the optimal rangefor the analytical enzyme, particularly when dilution of the fluid isnot practicable. An additional element of the invention is the use ofcompetitive inhibitors of the relevant analytical enzyme in order toreduce its sensitivity to the analyte, thereby permitting measurement ofthe latter at a higher concentration. This is especially useful, forexample, where selective binding agents are not readily available or areunacceptably expensive.

Another feature of the invention is that selective binding agents areemployed to reduce the free concentrations of interfering ions to levelswhere interference is no longer significant. Use is also made of thefact that a competitive inhibitor may compete more effectively withinterfering ions than with the analyte, thereby increasing thesensitivity of the enzyme to the analyte with respect to the interferingion.

An important element is the choice of optimal reaction conditions,including the selection of an appropriate isoenzyme, such that thestimulatory or inhibitory effects of the analyte are substantiallygreater that those of the interfering ions. In addition, the action ofthe analyte and interfering ions on the activity of the analyticalenzyme should be additive so that if the concentration of interferingions is known, the concentration of the analyte can readily bedetermined by difference. Where an interfering ion is known to occur ata relatively constant concentration in the fluid under analysis,allowance can be made for this by including an appropriate concentrationof the interfering ion in standard (calibrating) solutions. Anothermethod for assaying such analytes is the use of a competitive bindingassay where the analyte displaces another ion from a binding agent andthe effects of the released ion on the activity of an appropriate enzymeis determined.

These general principles can best be illustrated in detail by showingtheir application to the determination of potassium, sodium, calcium,chloride and bicarbonate ions in plasma or serum. However, they areapplicable to a wide spectrum of ions, for example cations such asmagnesium, manganese, lithium, lead, zinc, copper, iron or other heavymetals. Examples of non-metallic ions that can be measured are protonsor ammonium. Substances such as urea that give rise to ammonium can alsobe determined.

Suitable Enzymes

Enzymes which may used can be for example (H. J. Evans et al. Ann. Rev.Plant Physiol. 17, 47, 1966):

Transferases like phosphorus-containing group-transferring transferases.Such a transferase may be pyruvate kinase. In place of pyruvate kinaseother kinases such as adenylate kinase or hexokinase, sensitive tomagnesium ion or manganous ion may be employed. Another transferase isacetate kinase (from E. coli). Another example is pyridoxal kinase frombrain which is sensitive to zinc ions.

Hydrolases like glycosidases, for example α or β-D-galactosidase (fromEscherichia coli), carboxypeptidase A (from bovine pancreas),collagenase (from Clostridium hystolicum), amylase (from saliva orpancreas) or phosphoglycollate phosphatase.

Also peptide hydrolases such as the cysteine or thiol dependentproteinases, specific examples of which are Calpain I and II (alsocalled calcium activated neutral protease) described by Sasaki et al. inJ. Biol. Chem. 259, 12489-12494, (1984). The latter enzymes can beisolated and purified from a variety of animal tissues such as: ratliver and kidney, human and porcine erythrocytes, bovine brain, andrabbit skeletal muscle according to the method of A. Kitahara et al. ,described in J. Biochem. 95, 1759-1766 (1984). A further example isdipeptidyl aminopeptidase I (E.C. 3.4.14.1, Cathepsin C), J. Ken McDonald, Bloch. Biophys. Res. Communication 24(5), 66,771f. Anothersource for the enzymes are proteins obtained by gene recombinationtechniques.

Oxidoreductases like glycerol dehydrogenase (from Enterobacteraerogenes), acetaldehydrogenase (from yeast) or tyrosinase (catecholoxidase).

Lyases like aldolase (from yeast) or carbonic anhydrase (from bovineerythrocytes).

Other suitable enzymes are various enzymes from halophilic organisms.Another source for the enzymes are proteins obtained by generecombination techniques.

Selective Binding Agents: A wide variety of binding agents are availablefor the binding of analytes or interfering ions. Such binding or maskingsubstances are cryptands, coronands, crown ethers, podands, spherands,hemispnerands, calix arens and combinations thereof, natural occurringionophores, for example antibiotics, cyclic peptides like valinomycin,complexones and chelating agents, for example iminodiacetic acid, EDTA,nitrotriacetic acid and derivatives thereof. Such compounds aredescribed in Kontakte (Merck), 1977, No. 1, p. 11 ff and p. 29 ff;Kontakte (Merck), 1977, No. 2, p. 16 ff; Konakte (Merck), 1977, No. 3,p. 36 ff; Phase Transfer Catalysts, Properties and Applications(Merck-Schuchardt) 1987, Thermodynamic and Kinetic Data forCation-Macrocycle Interaction; R. M. Izatt et al., Chemical Reviews85,271-339 (1985); Data for Biochemical Research, 1986, R. M. C. Dawsonet al., Eds., 3rd edit., 399-415 (Clarendon Press) Oxford; F. Vo/ gtleet al., Chem. Macrocycles, Springer Verlag, N.Y., 1985; G. W. Gokel etal., Eds., Macrocyclic Polyether Synthesis, Springer Verlag, N.Y., 1982; M. Hiraoka, Ed., Crown Compounds, Elsevier, Amsterdam, Oxford, N.Y.,1982; J. M. Lehn et al., J.Am. Chem. Soc. 97, 6700-6707 (1975); G.Schwarzenbach et al., Helv.Chim. Acta 28, 828 (1945); S. F. A. Kettle,Koordinationsverbindungen, Taschentext 3, Verlag Chemie,Weinheim/Bergstr. 1972; A. B. Martell et al., Die Chemie derMetallchelatverhindungen, Verlag Chemie, Weinheim/Bergstr. 1958; M.Becke-Goehring et al., Komplexchemie, Springer Verlag, 1970; F. Kober,Grundlagen der Komplexchemie, Otto Salle Verlag, Frankfurt/Main 1979; G.Schwarzenbach et al;, Helv.Chim.Acta 31, 1029 (1948); R. G. Pearson etal., Science 151, 172 (1966).

Examples of chelators capable of binding multivalent ions, in particularbivalent cations are ethyl eneglycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid (referred to as EGTA) and(ethylenedinitrilo) tetraacetic acid (EDTA).

While many binding agents exist that can bind multivalent ions, e.g.EDTA and its derivatives, agents which bind monovalent ions are lesscommon. Tetraphenylboron binds potassium ions. However, a group ofcompounds with wider possibilities are cryptands which are examples ofreagents that can selectively bind monovalent cations in aqueoussolutions (R. M. Izatt et al., Chem. Reviews 85, 271-339). Specialexamples for cryptands are the Kryptofix® compounds of Merck-Schuchardt,for example:

4,7,13,16,21-Pentaoxa-1,10-diazabicyclo 8.8.5!-tricosan, Kryptofix® 221,page 438, Merck-Schuchardt catalogue, dated 1987/88, no. 810646 (K221).4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan, Kryptofix®222, page 438, Merck-Schuchardt catalogue, dated 1987/1988, no. 810647(K 222).

As masking compounds for the elimination of interfering artions thefollowing classes of substances may potentially be used:anioncryptandes, heterocyclophanes, catapinands and inorganic metalcomplexes or insoluble salts. Special examples of anion complexingcompounds are described in the literature, e.g. azamono- orazapolycycles, macrocyclic quarternary tetrahedron compounds,macrocyclic bis -metal-complexes, macrocycles with covalentlyincorporated lewis acid centers, protonated- or alkylated quarternarycryptands or catapinands (F. P. Schmidtchen, Nachrichten Chem. Techn.lab. 36(1), 1988, S. 8f; B. Graf, J. Amer. Chem. Soc. 98 (20), 1976,6403f; C. H. Park, J. Amer. Chem. Soc. 90 (9), 1968, 2431f) as well ase.g. the hexachloro complex of Fe (III) or silver nitrate.

Function of Binding Agents: These binding agents are used for thefollowing purposes:

1. The selective binding of interfering ions.

2. To reduce the concentration of the analytes to optimal measuringlevels, if dilution of the sample is not feasible.

3. An embodiment of the invention is a process, wherein the bindingagent is present and forms a complex with "indicator" ions, from whichcomplex the indicator ions are displaced stoichiometrically by theanalyte ions, and wherein the influence of the displaced indicator ionson the activity of an enzyme is assayed, thereby giving an indirectmeasure of the concentration of analyte ions. For example in such aprocess the enzyme is pyruvate kinase, the indicator ions are potassium,the binding agent is Kryptofix® 221 and the ion to be determined issodium; or the enzyme is a kinase, the indicator ion is Mg²⁺, thebinding agent is a chelating agent, e.g. EDTA, and the ion is a metal orthe enzyme is pyridoxal kinase, the indicator ion is Zn²⁺ the bindingagent is Kryptofix® 221, and the ion is a heavy metal; or the enzyme isα-amylase, the binding agent is Ag or Hg and the ion to be determined ischloride; or the enzyme is collagenase the binding agent is a chelatingagent such as EDTA and the ion to be determined is calcium.

Fluids for Analysis: The biological fluids in which the measurement ofanalytes is made are blood, serum, plasma, sweat, transudates orexudates or urine for example. Other examples of fluids are tap water orextracts of foodstuffs or fruits or fermented liquids such as wine.

Application of General Principles to the Determination of Potassium andSodium Ions

The essential requirements for a satisfactory method for thedetermination of potassium ions in serum or plasma, on the basis of thesensitivity of pyruvate kinase to potassium ions, is the overcoming ofthe interference by sodiums and ammonium ions. According to the generalprinciples embodied in this invention this can be achieved by one ormore of the following procedures:

1. The selective binding of sodium ions with a suit, able binding agent,for example Kryptofix® 221.

2. The selection of Bacillus stearothermophilus, rather than rabbitmuscle, as the source of pyruvate kinase since the bacterial enzyme hasa sensitivity for potassium ions in relation to sodium ions twice asgreat as the muscle enzyme.

3. Inclusion of ions which are competitive inhibitors of the sensitiveindicator enzyme in the assay, for example the use of lithium ions tocompete with sodium ions and potassium ions.

4. Enzymatic removal of ammonium ions.

Since lithium ions are less effective as a competitor against potassiumions, the net effect is to increase the relative sensitivity of pyruvatekinase towards potassium ions a further 50% as compared with sodiumions. Moreover, in the presence of lithium ions the effects of potassiumand sodium ions on the activity of pyruvate kinase become additive,rather than co-operative. This allows the possibility of measurement ofthe concentration of either potassium or sodium ions, provided that theconcentration of the other ions is known.

By the use of procedure 2 and 3 in the absence of a binding agent it ispossible to obtain a relative sensitivity of pyruvate kinase forpotassium versus sodium ions in the order of 100:1. This means that evenat extremely abnormal sodium ion concentrations of either 110 or 170mmol/l, the error in measured potassium ion concentration will notexceed 0.3 mmol/l relative to a normal plasma sodium ion concentrationof 140 mmol/l (Example A). This is not regarded as sufficiently accuratefor many clinical purposes. However, if the true concentration of sodiumions in the plasma is known, accurate measurement of potassium ions downto ±0.05 mmol/l is feasible (Example B). If procedures 1-3 are combinedand a binding agent, e.g. Kryptofix® 221 is included, the relativesensitivity of pyruvate kinase for potassium ions with respect to sodiumions can be increased to <500:1. Under these circumstances it is notnecessary to know the sodium ion concentration to determine the plasmapotassium ion concentration down to 0.05 mmol/l (Example C).

These methods for potassium ion determination demonstrate theapplicability of the general principles embodied in the invention inregard to reduction of interferences. On the other hand the measurementof sodium ions in serum or plasma best illustrates the application ofthese principles in regulating effective analyte concentration. Onemeans of measuring sodium ions as embodied in this invention is to usean enzyme whose activity is sensitive to sodium ions. An example of suchan enzyme is β-galactosidase (Kuby et al., J. Am. Chem. Soc. 75, 890,1953). However, the range of sodium ion concentration to which thisenzyme is most sensitive is much lower than can conveniently be obtainedin a plasma sample, without a dilution step.

In keeping with the principles embodied in this invention the followingprocedures are employed to lower the effective sodium ion concentrationto optimal levels when dilution of the sample is not feasible.

1. Use of a sodium ion binding agent such as Kryptofix® 221.

2. Use of lithium ions as a competitive inhibitor of β-galactosidase,thereby decreasing the sensitivity of the enzyme to sodium ions.

The combination of procedures 1 and 2 readily allows the determinationof sodium ions in plasma or serum, using β-galactosidase, and the amountof binding agent can be manipulated to minimise the signal for sodiumion concentrations below 110 mmol/l while enhancing the signal in theusual analytical range (110-170 mmol/l) (Example D).

Sodium ions may also be measured by means of pyruvate kinase, providedthat conditions are chosen whereby the stimulation of enzyme activity bypotassium ions is reduced, and a potassium ion-binding agent, e.g.Kryptofix® 222, is included in the reaction mixture (Example E).

In another method of determining plasma sodium ion concentration, thesodium ions are allowed to displace potassium ions from Kryptofix® 221,the released potassium ions stimulating the activity of pyruvate kinasein proportion to the plasma sodium ion concentration (Example F).

Other embodiments of the invention are compositions and reagents for thedetermination of ions in biological and non-biological fluids.

The reagent according to the present invention can be present indissolved or dry form. It can be present impregnated on an appropriatecarrier. A diagnostic agent in the form of a test strip can be producedby impregnating a carrier material, preferably filter paper, celluloseor synthetic fibre fleece, with solutions of the necessary reagentsconventionally used for the production of test strip in readily volatilesolvents, such as acetone. This can take place in one or moreimpregnation steps. The finished test papers can be used as such orstuck in known manner on to handles or preferably sealed betweensynthetic resins and fine meshes.

The embodiments of the invention are described hereunder in some detailbut it will be seen that the invention need not necessarily be limitedin any one or in a combination of the details described, and inparticular, mechanical or chemical variations can be utilized besidesthose described in this embodiment.

Detailed Description of Analytical Methods for Potassium Ions

This section describes in more detail methods for potassium iondetermination embodying the principles described in this invention. Forthe determination of potassium ions, a fluid, for example blood plasma,is incubated with a buffered mixture containing adenosine diphosphate(ADP), phosphoenolpyruvate (PEP), reduced nicotinamine adeninedinucleotide (NADH), pyruvate kinase (PK) and lactate dehydrogenase(LDH). The formation of pyruvate, and subsequently lactate in thismixture, in reactions catalysed by PK and LDH, is entirely dependent onthe presence of appropriate cations, in the absence of which PK isvirtually inactive. NADH absorbs strongly at 340 nm, whereas AND doesnot.

Under the conditions chosen for analysis which include the presence ofmanganese ions which are required by the bacterial PK, the rate of NADHoxidation is proportional to the concentration of potassium ions (seeExamples A-C).

Under these conditions the following reactions take place: ##STR1##

The rate of reaction (1) is determined by the concentration of potassiumions present in the system, and this in turn limits the rate of reaction(2). There are several ways in which the rates of these reactions can bemeasured. A standard approach is the spectrophotometric measurement ofthe rate of disappearance of NADH in reaction (2). NADH absorbs stronglyat 340 nm, whereas NAD does not. Accordingly, the fall in absorbance ofthe reaction mixture at 340 nm (or an alternative wavelength) provides adirect measure of the rate of the reaction and from this theconcentration of potassium ions present in the mixture can be derived.Alternatively, advantage can be taken of the fact that both reaction (1)and (2) consume H⁺, thus lowering the proton concentration of thereaction mixture. The rate of fall in proton concentration can bemeasured with a pH meter, or by means of a titration procedure. In theselatter cases the concentration of buffer employed will be much less thanin the spectrophotometric technique. Other equipment such asfluorimeters or luminometers can be used to monitor the activity ofpyruvate kinase.

There are a number of other methods of detecting the accumulation ofpyruvate associated with PK activity. These include any method formeasuring the inorganic phosphate or oxygen consumed or the hydrogenperoxide; acetyl phosphate or carbon dioxide generated by the enzymaticaction of pyruvate oxidase; the formation of the hydrazone with2,4-dinitrophenylhydrazine; the measurement of the reactants or productsof the enyzmatic action of pyruvate carboxylase; pyruvate decarboxylaseor pyruvate dehydrogenase; the use of flavine coupled systems; andisotopic methods for measuring minute concentrations of substrates (M.N. Berry et al., Analytical Biochem. 118, 344-352 (1981)).

In a survey of 200 serum samples good agreement has been obtained withother methods such as flame photometric or ion-selective electrodemeasurements. A significant interference with the method is ammoniumions which are generally present in serum or accumulate on standing. Thepossibilly of ammonium ion interference can be completely avoided byincluding α-ketoglutarate (KG) and glutamaro dehydrogenase (GDH) in thereaction mixture. Ammonium ions are removed in a preincubation accordingto the reaction:

NH₄ ⁺ +glutamate+NADH→glutamate+NAD⁺

In solutions such as urine in which the ammonium ion content may be higha coupled reaction can be used.:

NH₄ ⁺ +KG+NADPH→glutamate+NADP

glucose-6-P+NADP, →6-phosphogluconate+NADPH

The coupled method employs glucose-6-phosphate dehydrogenase. Providedthat the added glucose-6-P and KG are in excess of any ammonium ionspresent, all ammonium ions will be removed while preserving the NADH inthe reagent.

Typical concentration ranges of the main reagents for the enzymatoicdetermination at 37° C. of potassium ions using a 10 μl sample of plasmaor serum are:

    ______________________________________    PK (B. stearothermophilus)                    50 U/l      to    10 000 U/l    PEP (neutralized Tris salt)                    0.3 mmol/l  to    30 mmol/l    Kryptofix ® 221                    0 mmol/l    to    30 mmol/l    NADH            0.01 mmol/l to    0.8 mmol/l    buffer, pH 7-8  50 mmol/l   to    500 mmol/l    Mn.sup.2+  or Mg.sup.2+                    1 mmol/l    to    10 mmol/l    LiCl            2 mmol/l    to    100 mmol/l    ADP (free acid) 0.5 mmol/l  to    10 mmol/l    LDH (assayed at 25° C.)                    5 000 U/l   to    100 000 U/l    Serum albumin   0 g/l       to    5 g/l    GDH (assayed at 25° C.)                    2 500 U/l   to    20 000 U/l    KG (free acid)  1 mmol/l    to    10 mmol/l    ______________________________________

Another example sensitive to potassium ions is glycerol dehydrogenase(E. C. C. Linet al., B 235, 1820, 1960). Typical concentration ranges ofthe main reagents for the enzymatic determination at 37° of potassiumions using glycerol dehydrogenase are:

    ______________________________________    Glycerol dehydrogenase                     50 U/l    to    1000 U/l    Glycerol         0.3 mol/l to    3 mol/l    Kryptofix ® 221                     0 mmol/l  to    30 mmol/l    NAD              0.1 mmol/l                               to    5.0 mmol/l    buffer, pH 9     20 mmol/l to    500 mmol/l    Serum albumin    0 g/l     to    5 g/l    GDH (assayed at 25° C.)                     2 500 U/l to    20 000 U/l    KG (free acid)   1 mmol/l  to    10 mmol/l    ______________________________________

Another enzyme sensitive to potassium ions is acetaldehyde dehydrogenase(S. Black, Arch. Biochem. Biophys. 34, 86, 1951). Typical concentrationranges of the main reagents for the enzymatic determination at 37° C. ofpotassium ions using acetaldehyde dehydrogenase are:

    ______________________________________    Acetaldehyde dehydrogenase                     50 U/l     to    10 000 U/l    Glycolaldehyde   0.3 mmol/l to    30 mmol/l    Kryptofix ® 221                     0 mmol/l   to    30 mmol/l    NAD              0.05 mmol/l                                to    2.0 mmol/l    buffer, pH 7-8   50 mmol/l  to    500 mmol/l    Dithiothreitol   0.1 mmol/l to    2 mmol/l    Serum albumin    0 g/l      to    5 g/l    GDH (assayed at 25° C.)                     2 500 U/l  to    20 000 U/l    KG (free acid)   1 mmol/l   to    10 mmol/l    ______________________________________

Acetaldehyde (0.02 mmol/l to 1 mmol/l) may be substituted forglycolaldehyde.

Acetaldenyde dehydrogenase also exhibits esterase activity so thatpotassium ion concentration can be determined by monitoring the releaseof 4-nitrophenol from 4-nitrophenyl acetate. Typical concentrationranges of the main reagents for the enzymatic determination at 37° C. ofpotassium ions based on the esterase activity of acetaldehydedehydrogenase are:

    ______________________________________    Acetaldehyde dehydrogenase                     50 U/l     to    10 000 U/l    4-nitrophenyl acetate                     0.1 mmol/l to    2 mmol/l    Kryptofix ® 221                     0 mmol/l   to    30 mmol/l    NADH             0.001 mmol/l                                to    0.1 mmol/l    buffer, pH 7-8   50 mmol/l  to    500 mmol/l    Dithiothreitol   0.1 mmol/l to    2.0 mmol/l    Serum albumin    0 g/l      to    5 g/l    GDH (assayed at 25° C.)                     2 500 U/l  to    20 000 U/l    KG (free acid)   1 mmol/l   to    10 mmol/l    ______________________________________

Determination of Sodium Ions

In principle the measurement of sodium ions, using PK, is similar tothat of potassium ions, However, certain key differences exist. In thefirst instance PK is some 40-100 times more sensitive to potassium ionthan it is to sodium ion, depending on incubation conditions asdescribed above. Hence even though sodium ions occur in the plasma at aconcentration some 30 times that of potassium ions, the latter caninterfere with sodium ion measurement.

According to the general principles espoused in this invention, lithiumions are omitted, PK from rabbit muscle is preferred to the bacterialenzyme, and magnesium ions may be substituted for manganese. In onemethod the potassium ions are specifically bound with Kryptofix® 222 andthe effects of sodium ions on PK activity measured directly (Example E).

Typical concentration ranges of the main reagents for the enzymaticdetermination at 37° C. of sodium ions using pyruvate kinase are:

    ______________________________________    PK (rabbit muscle)                    50 U/l      to    10 000 U/l    PEP (neutralized Tris salt)                    0.3 mmol/l  to    30 mmol/l    Kryptofix ® 222                    0.4 mmol/l  to    4 mmol/l    NADH            0.01 mmol/l to    0.8 mmol/l    buffer, pH 7-9  50 mmol/l   to    500 mmol/l    Mg.sup.2+       1 mmol/l    to    10 mmol/l    ADP (free acid) 0.5 mmol/l  to    10 mmol/l    LDH (assayed at 25° C.)                    5 000 mmol/l                                to    100 000 U/l    Serum albumin   0 mmol/l    to    5 mmol/l    GDH (assayed at 25° C.)                    2 500 U/l   to    20 000 U/l    KG (free acid)  1 mmol/l    to    10 mmol/l    ______________________________________

In another method the sodium ions are allowed to displace potassium ionsfrom Kryptofix® 221, the released potassium ions stimulating theactivity of PK to a degree dependent on the sodium ion concentration(Example F).

Typical concentration ranges of the main reagents for the enzymaticdetermination at 37° C. of sodium ions using pyruvate kinase to measurethe displacement of potassium ions from Kryptofix® 221 are:

    ______________________________________    PK (rabbit muscle)                    50 U/l      to    10 000 U/l    PEP (neutralized Tris salt                    0.3 mmol/l  to    30 mmol/l    Kryptofix ® 221                    1 mmol/l    to    10 mmol/l    NADH            0.01 mmol/l to    0.8 mmol/l    buffer, pH 9-10 50 mmol/l   to    500 mmol/l    Mg.sup.2+       1 mmol/l    to    10 mmol/l    ADP (free acid) 0.5 mmol/l  to    10 mmol/l    LDH (assayed at 25° C.)                    5 000 mmol/l                                to    100 000 U/l    KCl             2 mmol/l    to    10 mmol/l    Serum albumin   0 g/l       to    5 g/l    GDH (assayed at 25° C.)                    2 500 U/l   to    20 000 U/l    KG (free acid)  1 mmol/l    to    10 mmol/l    ______________________________________

A more accurate and precise embodiment for determining uses a sodium iondependent enzyme such as β-galactosidase. (Example D).

Blood plasma is incubated with a buffered mixture containing2-nitrophenyl-β-D-galactopyranoside (NPG) and the enzymeβ-D-galactoside. The reaction catalyzed by β-D-galactosidase isdependent on the presence of sodium ions and the rate of activity is ameasure of sodium ion concentration. A key feature of this method is theuse of an appropriate amount of sodium ion selective binding agent (e.g.Kryptofix® 221) for measurements in serum or other biological fluidswhere the sodium ion concentration may exceed 100 mmol/l, to reducesodium ion concentration so that the enzyme is most sensitive to smallchanges in sodium ion concentration in the usual analytical range(110-170 mmol/l). Under the conditions chosen for analysis, whichinclude the presence of moderately high concentrations of magnesium andlithium ions, the rate of 2-nitrophenol and galactose formation isvirtually proportional to the concentration of sodium ions beingmeasured. Magnesium ions are required for optimal β-galactosidaseactivity. Lithium ions are competitive with sodium ions and thereforeraise the K_(m) of the enzyme for sodium ions.

As substrate for β-D-galactosidase many other compounds are suitable.Quite generally, the galactosidase-containing sample is mixed with anappropriate β-D-galactosidase substrate, the substrate being split bythe enzyme, one of the fission products then being detected in anappropriate manner. Either the glycone liberated by action of the enzymeor the aglycone can be measured. As a rule, the latter is determined. Assubstrate, the natural substrate lactose can be used, but especiallyadvantageous is use of a chromogenic galactoside. Thus, in Biochem. Z.,333, 209 (1960), there are described phenyl-β-D-galactoside, as well assome further derivatives substituted on the aromatic ring, for example,2-nitrophenyl-β-D-galactoside (NPG) and 3-nitrophenyl-β-D-galactoside,as substrates of β-D-galactosidase. The phenols liberated by hydrolysisare determined photometrically in the UV range or, in the case of thenitrophenols, in the short-wave visible wavelength range. An oxidativecoupling with aminoantipyrine can also follow as indicator reaction (seeAnalytical Biochem. 40, 281 (1971)). Other substrates are described inthe German Offenlegungsschrift 33 45 748 and the GermanOffenlegungsschrift 34 11 574.

Typical concentration ranges of the main reagents for the enzymaticdetermination at 37° C. of sodium ions using a 10 μl sample of plasma orserum are:

    ______________________________________    D-galactosidase 25 U/l     to    7 500 U/l    NPG             0.25 mmol/l                               to    5 mmol/l    Kryptofix ® 221                    0 mmol/l   to    10 mmol/l    NADH            200 mmol/l to    500 mmol/l    buffer, pH 7-9.5                    200 mmol/l to    500 mmol/l    Mg.sup.2+       0.01 mmol/l                               to    10 mmol/l    EGTA (Lithium salt)                    0 mmol/l   to    20 mmol/l    Serum albumin   0 g/l      to    5 g/l    ______________________________________

EGTA mans Ethylenbis (oxyethylennitrilo)-tetraacetic acid. It will alsobe feasible to perform the analysis of sodium and potassium ionsenzymatically in the same cuvette in the form of a twin test (seeExample G).

Determination of Calcium Ions

For the measurement of calcium ions, a sample of blood plasma (or otherbody fluid) is incubated with a buffered mixture containing the peptidesubstrate succinyl--leucine--methionine--p-nitroanilide and the enzymeCalpain I. The reaction catalyzed by Calpain I is dependent on thepresence of calcium ions and the rate of activity is a measure of thecalcium ion concentration. A key feature of the method is the use ofchelators capable of specifically binding calcium ions in order to lowertheir concentration to a range over which the enzyme is most sensitive.Under the conditions chosen for analysis, which include the presence ofL-cysteine and 2-mercaptoethanol, the rate of p-nitroaniline formationis virtually proportional to the concentration of calcium beingmeasured.

Preferred peptide substrates can be described by the general formula

    R - P.sub.n - P.sub.2 - P.sub.1 - X

whereby R represents acetyl, benzoyl, carbobenzoxy, succinyl,tert-butoxy carbonyl or 3-(2-furyl)acryloyl; P_(n) -P₂ -P₁ represents apeptide chain of a least 2 residues with a preference for Tyr, Met, Lysor Arg in the P₁ position and a Leu or Val residue in the P₂ position;and X represents a chromogenic or fluorogenic group which is liberatedby the action of the enzyme to yield a detectable change in color orfluorescence. X can be a nitrophenyl, naphthyl or thiobenzyl ester aswell as a nitroaniline, naphthylamine or methylcoumarin group eitherwith or without further substituents on the aromatic ring. Some suitablepeptide derivatives have also been described by T. Sasaki et al. in J.Biol. Chem. 259, 12489-12494, examples are succinyl--Leu--Met--MCA(MCA=4-methylcoumarin-7-amide), succinyl--Leu--Tyr--MCA,succinyl--Leu--Leu--Val--Tyr--MCA and tert-butoxycarbonyl--Val--Leu--Lys--MCA. Further synthetic substrates are describedin Bergmeyer HU (Ed) Methods of Enzymatic Analysis, 3rd Edition, Volume5, p. 84-85 (1984).

The concentration ranges of the compounds for such a determinationmethod and reagents are:

    ______________________________________    Calpain I         1 000 U/l  to    40 000 U/l*    Suc--Leu--Met-p-nitroanilide                      1 mmol/l   to    20 mmol/l    Chelator          0.01 mmol/l                                 to    1 mmol/    L-Cysteine        1 mmol/l   to    10 mmol/l    2-Mercaptoethanol 1 mmol/l   to    10 mmol/l    Buffer Imidazole-HCl                      10 mmol/l  to    100 mmol/l    pH                6-8 (preferred range 7-7.5)    ______________________________________

The asterisk * means that unit is defined as the quantity of enzymewhich increases the absorbance at 750 nm by 1.0 after 30 min ofincubation of 30° C. with casein as substrate (N. Yoshimura et al., J.Biol. Chem. 258, 8883-8889, (1983)).

Any buffer having a pK in the required pH range with a negligiblebinding capacity for calcium may be used in the assay. Many of theGood-type buffers (N. E. Good et al., Biochem. 5, 467-477 (1966)) suchas Tris, HEPES, MOPSO, BES, TES and imidazole fulfill these requirements(see example H). Collagenase may be used instead of Calpain I, andassayed fluorometrically at pH 6.5-7.5 with L-isoleucyl-L-analylglycylesthylester, 0.02 mmol/l to 0.2 mmol/l.

Determination of Chloride Ions

For the determination of chloride in blood, plasma is incubated with abuffered mixture containing 0.01 mol/l cysteamine, 4 mmol/lGly--Phe--p-nitroanilide and 0.02 U/ml Cathepsin C. The formation ofp-nitroaniline is entirely dependent on the presence of the chlorideanions. Selective binding agents may be added in addition in order toeliminate interference by bromide ions or to decrease the activity ofchloride ions so as to adjust their concentration to the optimal rangeof the enzyme. Under the conditions chosen for analysis (see example 5)the rate of formation of p-nitroaniline: ##STR2## is proportional to theconcentration of chloride ions in the sample. In this example the rateof the reaction is determined by measurement of the increase ofabsorption at 405 nm.

The concentration ranges of the compounds of such a determination methodare:

    ______________________________________    citrate buffer    0.01-0.2 mol/l    pH                4-7 (preferred: 5.0-5.5)    cysteamine        1-20 mmol/l    gly--arg-p-nitroanilide                      1-20 mmol/l    cathepsin C       2-100 mU/ml    ______________________________________

Any buffer having a pK in the required pH-range and a negligiblechloride concentration may be used in the assay. Examples of enzymesfrom all of the categories listed above (transferases, hydrolases,oxidoreductases and lyases) have been shown in the literature to have achloride dependency, especially if the origin of these enzymes is fromhalophilic organisms. The chloride dependency of Enzymes of thepeptidase type has been extensively described. For examplesdipeptidylpeptidase I (Cathepsin C), ED 3.4.14.1 (J. Ken McDonald,Bioch. Bioph. Res. Communication, 24 (5) 1966, 771f) ordipeptidylpeptidase II EC. 3.4.14.3 (J. Ken McDonald, Journal of Biol.Chem. 241 (7) 1966, 1494f), both catalyzing the hydrolysis ofoligopeptide derivatives from the amino terminal end. Another example isthe angiotensin converting enzyme EC 3.4.15.1 which is adipeptidylcarboxypeptidase that catalyses the hydrolytiqrelease ofdipeptides from the carboxyl terminus of a broad range of oligopeptides(P. Bunning et al., Bioch. 26, 1987, 3374f; R. Shapiro et al., Bioch.22, 1983, 3850f).

Instead of Gly--Arg--p-nitroanilide different other dipeptide- oroligopeptide substrates may be used. Preferred peptide substrates can bedescribed by the general formula

    R-P.sub.n -P.sub.1 -X

whereby for the dipeptidyl peptidase enzymes R=H and P_(n) -P₁represents a peptide chain of at least 2 residues. X represents achromogenic or fluorogenic group which is liberated by the action of theenzyme to yield a detectable change in color or fluorescence. X can be anitrophenyl-, naphthyl- or thiobenzylester as well as a nitroaniline,naphthylamine or methylcoumarin group either with or without furthersubstituents on the aromatic ring. In the case ofdipetidylcarboxypeptidase enzymes X represents the amino terminal and ofthe peptide chain and R is a chromogenic or fluorogenic group which isliberated by the action of the genzyme. R can be a N-2-furanacryloyl- orbenzoyl-group either with or without further substituents (see exampleI).

As a further example of the enzymatic determination of chloride ione(Example J), plasma is incubated with a buffered mixture containing4,6-ethylidene (G₇)-p-nitrophenyl (G₁)- D-maltoheptaoside(4,6-ethylidene-G₇ PNP) (5 mmol/l), α-amylase (0.60 U/ml) andα-glucosidase (=30 U/ml). The formation of p-nitrophenol is entirelydependent on the presence of chloride anions, once again using eitherpredilution, small sample volumes or selective binding agents to adjustchloride ion concentration to the optimal range of the enzyme. The testprinciple is summarized below, and the rate of the reaction isdetermined by the measurement of the increase in absorption at 405 nm.##STR3## 2-ethylidene-G₅ +2 G₂ PNP+2 ethylidene-G₄ +2 G₃PNP+ethylidene-G₃ +G₄ PNP ##STR4##

The concentration ranges of compounds used for such a determination are:

    ______________________________________    Hepes or alternative chloride free                        0.01-0.5   mmol/l    buffer    pH                  6.5-7.5    α-amylase     60-6000    U/l    α-glucosidase 3000-300 000                                   U/l    4,6-ethylidene-G.sub.7 PNP                        0.5-10     mmol/l    ______________________________________

Assay variations discussed above for Cathepsin C (Example I) also applyto Example J.

Determination of Heavy Metal Ions

The metals bind tightly to cryptands such as Kryptofix® 221 andKryptofix® 222. They will therefore displace other metals that are moreloosely bound. An example of a metal ion readily displaced is zinc. Zincions are present in very low concentration in plasma. Thus it isfeasible to add zinc ions complexed to K 221 to buffered serum mixture.If a heavy metal is present the zinc ions will be liverated and theirpresence can be detected by stimulation of pyridoxal kinase (from sheepbrain) an enzyme which is highly sensitive to zinc ions. Many othersimilar competitive binding assays are feasible, and those described aregiven by way of example and not of limitation.

Determination of Bicarbonate Ions

Bicarbonate ions can be measured using a variation of the principlesembodied in the invention. Many ligands, e.g. the cryptands, are pHsensitive, and this property can be exploited to measure bicarbonate.Essentially, advantage is taken of the ability of bicarbonate toneutralize protons. It can be shown that the pH of a very lightlybuffered serum sample, to which hydrochloric acid has been added willvary as a function of the bicarbonate concentration. The final pH isdetected by the amount of free sodium ions (as detected with5-galactosidase) present in the presence of a pH-sensitive ion-bindingagent such as Kryptofix® 221, and this is a function of the originalbicarbonate concentration. In essence, serum is acidfied to pH 4.5approx, with an equal volume of HCl (75 mmol/l) to convert allbicarbonate to hydroxyl ions, and then reacted with an assay system atpH 7.5-7.8, using a dilute Tris buffer (5 mmol/l) which incorporates anion-selective enzyme, such as β-galactosidase and appropriatepH-sensitive ion binding agent. The sodium ion concentration of thesample must be known to obtain accurate results since a correction isnecessary based on the quantity of sodium ions in the sample. The methodis substantially more sensitive than procedures using chromogenicindicators as pH detectors (Example K).

Typical concentration ranges of the main reagents for the enzymaticdetermination at 37° C. of bicarbonate ions using a 10 μl sample ofplasma or serum are:

    ______________________________________    D-galactosidase 250 U/l    to    7500 U/l    NPG             0.25 mmol/l                               to    5 mmol/l    Kryptofix ® 0.2 mmol/l to    5 mmol/l    buffer, pH 7.5-7.8                    1 mmol/l   to    10 mmol/l    Mg.sup.2+       0.01 mmol/l                               to    10 mmol/l    EGTA (Li salt)  0.1 mmol/l to    5 mmol/l    Serum albumin   0 g/l      to    5 g/l    ______________________________________

The need to correct for sodium ion concentration can be avoided by usingan enzyme (e.g. pyridoxal kinase) sensitive to trace metals (e.g. zincions) normally present in plasma in ulcromolar concentration. Providedthat the binding of the trace metal to its binding agent is pH sensitiveand possesses a similar affinity to sodium ions for Kryptofix® 221,bicarbonate ions can be measured by including the zinc ions in thereaction mixture in concentrations sufficiently in excess of those thatcan be encountered in plasma. Hence endogenous zinc ions will notinterfere.

The methods as described above are simple, very rapid, accurate andprecise and can be performed with inexpensive apparatus. The laboratoryhazard of inflammable gases can be avoided as can the many problemsassociated with ion-selective electrodes. The method can be adapted foruse with large equipment performing multiple analyses, yet can also beemployed with inexpensive stand-alone instruments for emergency useclose to the bedside. The packaging of the method in kit form isstraightforward. Moreover, the determination of potassium and sodium ionconcentration can be performed sequentially in the same cuvette (ExampleG). It is also intended that the method be useful for Doctors' officeswith a machine employing dry chemistries. Although these methods havebeen developed using automatic spectrometers, they are readily adaptableto automated or manual laboratory equipment such as fluorimeters,luminometers, isotope counters etc.

The present invention will now be described in more detail withreference to the following examples, on the basis of a serum or plasmasample of 10 μl. These examples are given by way of illustration and notof limitation.

In the following Examples a small volume of sample (10 μl except whereotherwise indicated in the case of serum or plasma) is mixed withReagent 1, containing buffered substrate and certain cofactors, andincubated for a period of time, generally 0.1-5 min. Absorbance readingsare normally taken at regular intervals during this incubation period.Reagent 2, containing the indicator enzyme is then added and thereaction rate monitored. In some Examples, Reagent 1 contains theindicator enzyme and Reagent 2 the appropriate substrate. The Examplesshow the final reaction mixture after the sample, Reagent 1 and Reagent2 have been mixed.

EXAMPLE A Measurement of Potassium Ion Concentration Using PyruvateKinase Without a Sodium-Ion-Binding Agent, Sodium Ion ConcentrationUnknown

The final incubation mixture contains:

    ______________________________________    175     mmol/l   Tris-HCl buffer, pH 7.4    20      mmol/l   Li.sup.+   17 mmol/l LiOH, 3 mmol/l LiCl!    3.0     mmol/l   MnCl.sub.2    2.6     mmol/l   ADP (free acid)    2.9     mmol/l   PEP (neutralized tris salt)    0.4     mmol/l   NADH    17000   U/l      LDH (assayed at 25° C.)    890     U/l      PK from Bacillus stearothermophilus    4.0     mmol/l   KG    8600    U/l      GDH (in glycerol; assayed at 25° C.)    140     mg/l     Human serum albumin    ______________________________________

Potassium ion standards (calibrating solutions) contain 140 mmol/lsodium ions to compensate for the stimulatory effect of sodium ions,present in plasma, on pyruvate kinase.

EXAMPLE B Measurement of Potassium Ion Concentration Using PyruvateKinase, Without a Sodium Ion Binding Agent, Sodium Ion ConcentrationKnown

The incubation mixture and calibrating solution are the same as forExample A.

A correction may be made for the sodium ion concentration of the mixtureby adding (or subtracting) 0.1 mmol/l potassium for every 10 mmol/l thesodium ion concentration is below (or above) 140 mmol/l sodium ions.However, this correction should be verified by analysing aqueoussolutions containing known sodium and potassium concentrations.

EXAMPLE C Measurement of Potassium Ion Concentration Using PyruvateKinase in the Presence of a Sodium Ion-Binding Agent

As for Example B, but human serum albumin is omitted and the mediumcontains in addition 6 μmol of Kryptofix® 221 per assay. A pH of 7.8 isselected to minimise variations in displacement of sodium ions fromKryptofix® 221 due to the differing potassium ion content of individualspecimens.

EXAMPLE D Measurement of Sodium Ion Concentration Usingβ-D-galactosidase

Variation a: The final incubation mixture contains:

    ______________________________________    300     mmol/l       Tris HCl, pH 8.7 (37° C.)    4       mmol/l       Dithiothreitol    7.5     mmol/l       Magnesium sulphate    16      mmol/l       Lithium chloride    0.44    mmol/l       EGTA (lithium salt)    460     mg/l         Human Serum Albumin    760     U/l          β-Galactosidase    1.5     mmol/l       NPG    1.25    μmol/assay                         Kryptofix ® 221    ______________________________________

The reaction is monitored at 420 nm (or nearby) wavelength to determinethe rate of formation of free 2-nitrophenol and hence the concentrationof sodium ions in the original sample.

Variation b: An alternative approach compared with Variation a would beto reduce the sample concentration ten-fold by pre-dilution or to use asmall sample volume, in which case the cryptand could be omitted.

Variation c: Measurement in fluids of low sodium ion content (e.g. (20mmol/l) in which case the cryptand could be omitted.

EXAMPLE E Measurement of Sodium Ions with Pyruvate Kinase (DirectStimulation of Enzyme Activity by Sodium Ions, Under Conditions WhereSensitivity of Potassium Ions is Diminished)

Variation a: Incubation mixture contains for a 10 μl plasma sample:

    ______________________________________    300   mmol/l    Tris HCl, pH 8.7 (37° C.)    5     mmol/l    MgCl.sub.2    2.6   mmol/l    ADP (free acid)    2.9   mmol/l    PEP (neutralized Tris salt)    0.34  mmol/l    NADH    17 000          U/l       LDH (assayed at 25° C.)    2 000 U/l       PK (from rabbit muscle, assayed at 37° C.)    4     mmol/l    KG    8 600 U/l       GDH in glycerol (assayed at 25° C.)    1.25  μmol/assay                    Kryptofix ® 222    ______________________________________

Sodium ion calibrating solutions contain 4 mmol/l potassium tocompensate for the potassium activating effect of serum potassium ionson pyruvate kinase.

EXAMPLE F Measurement of Sodium Ions with Pyruvate Kinase (CompetitiveBinding Assay)--Potassium Ion Concentration Known

The final incubation mixture contains for a 10 μl plasma sample:

    ______________________________________    300   mmol/l    Glycine, pH 9.8    5     mmol/l    MgCl.sub.2    2.6   mmol/l    ADP (free acid)    2.9   mmol/l    PEP (neutralized Tris salt)    0.34  mmol/l    NADH    17 000          U/l       LDH (assayed at 25° C.)    890   U/l       PK from Rabbit muscle (assayed at 37° C.)    4     mmol/l    KG    8 500 U/l       GDH (assayed at 25° C.)    5     mmol/l    KCl    2.5   μmol/assay                    Kryptofix ® 221    ______________________________________

Potassium chloride is added to this reagent and displacedstoichiometrically from Kryptofix® 221 by sodium ions, thus allowing thesodium ion concentration of the specimen to be quantified.

EXAMPLE G Measurement of Potassium and Sodium Ion Concentration in theSame Cuvette (Twin-Test)

Sodium ion concentration is assayed first as in Example D except thatthe assay also contains:

    ______________________________________    2.6      mmol/l     ADP (free acid)    2.9      mmol/l     PEP (neutralized Tris salt)    0.4      mmol/l     NADH    4.0      mmol/l     KG    8 600    U/l        GDH    ______________________________________

Following the measurement of sodium ions by means of determination thereaction rate, the pH of the incubation mixture is lowered to pH 7.4with a hydrochloric acid liquot.

Then the following ingredients are added to achieve the finalconcentrations indicated:

    ______________________________________    17 000  U/l       LDH    890     U/l       PK from Bacillus stearothermophilus    3.0     mmol/l    MnCl.sub.2    20.0    mmol/l    LiCl    ______________________________________

The reaction rate may then be monitored at 340 nm but may also bemeasured at a slightly higher wavelength to minimise possibleinterference by the 2-nitrophenol liberated in the sodium ion indicatorreaction.

EXAMPLE H Measurement of Calcium Ion Concentration Using Calgain I

In this embodiment, a small sample of blood is centrifuged to obtainplasma. For the measurement of calcium ions the sample was incubatedwith a mixture containing 50 mmol/l imidazole-HCl buffer, pH 7.3, 5mmol/l L-cysteine, 2.5 mmol/l 2-mercaptoethanol, 0.1 mmol/l EGTA and 5mmol/l Suc--Leu--Met-p-nitroanilide at 30° C. The increase of absorbanceat 405 nm was monitored over at 5 min interval. The rate is proportionalto the calcium ion concentration of the sample.

EXAMPLE I Measurement of Chloride Ion Concentration Using Cathepsin C

In this embodiment a small sample of blood is centrifuged to obtainplasma (5 μl). For the measurement of chloride ion the incubationmixture contains 0.05 mol/l citrate buffer pH-5.0, 10 mmol/l cysteamineand 4 mmol/l gly--arg--p-nitroanilide and 0.01 U/ml cathepsin C. Thelatter has been dialyzed against 10 mmol/l sodium phosphate buffer (pH -6.8) and 43% (v/v) of glycerol in order to remove chloride ions.

EXAMPLE J Measurement of Chloride Ion Concentration Using Amylase

Inhibition mixture contains for a 5 μl plasma sample:

    ______________________________________    Hepes or alternative chloride free buffer                           100      mmol/l    pH                     7.1    α-Amylase        600      U/l    α-Glucosidase    30 000   U/l    4,6-Ethylidene-G.sub.7 PNP                           5        mmol/l    ______________________________________

The reaction is monitored at 405 nm (or nearby wavelength to determinethe rate of formation of free 4-nitrophenol and hence the concentrationof chloride ions in the original sample.

Example K Measurement of Bicarbonate Ion Concentration Usingβ-D-galactosidase

Variation a: The final incubation mixture contains:

    ______________________________________    5       mmol/l        Tris HCl, ph 7.8 (37° C.)    4       mmol/l        Dithiothreitol    7.5     mmol/l        Magnesium sulphate    16      mmol/l        Lithium chloride    0.44    mmol/l        EGTA (lithium salt)    460     mg/l          Human serum albumin    1500    U/l           β-Galactosidase    1.5     mmol/l        NPG    2.0     μmol/assay Kryptofix ® 221    ______________________________________

EGTA means Ethylenbis(oxethylennitrilo)-tetraacetic acid. The sample ispre-incubated with an equivalent volume of HCl (for plasma, 75 mmol/l)to reduce sample pH to 4.5. The reaction is then monitored at 420 nm (ornearby) wavelength to determine the rate of formation of free2-nitrophenol and hence to concentration of bicarbonate ions in theoriginal sample. A correction is made for sodium ion concentration inthe original specimen.

Variation b: Pyridoxal kinase, pyridoxal and zinc ions are substitutedfor β-galactosidase and NGP.

We claim:
 1. Process for the determination of the concentration ofpotassium ions in a fluid sample, comprising measuring the activity ofan enzyme whose activity is stimulated by potassium ions and is selectedfrom the group consisting of a transferase or an oxidoreductase in thepresence of a competitive inhibitor ion which is present in an amountsufficient to increase the ratio of the activity of the enzyme to theconcentration of free potassium ions in the fluid sample to within anoptimal range for measurement of the concentration of potassium ions inthe fluid sample, wherein the measured activity of said enzyme isproportional to the concentration of said potassium ions in the fluidsample.
 2. Process according to claim 1, wherein said fluid sample iswater.
 3. Process according to claim 1 wherein said fluid sample is abody fluid.
 4. Process according to claim 3, wherein said body fluid isselected from the group consisting of blood, serum, plasma, urine,sweat, exudates or transudates.
 5. Process according to claim 3, whereinthe transferase is a microbial transferase.
 6. Process according toclaim 5, wherein the microbial transferase is bacterial pyruvate kinase.7. Process according to claim 6, wherein the competitive inhibitor ionis a lithium ion.
 8. Process according to claim 3 wherein theoxidoreductase is acetaldehyde dehydrogenase or glycerol dehydrogenase.9. Process according to claim 8, wherein said competitive inhibitor ionis a lithium ion.
 10. Process according to claim 3, wherein saidcompetitive inhibitor ion is a lithium ion.
 11. Process for thedetermination of the concentration of potassium ions in a fluid sample,comprising measuring the activity of an enzyme whose activity isstimulated by potassium ions and is selected from the group consistingof a transferase or an oxidoreductase in the presence of a firstselective binding agent which binds to an interfering ion in the fluidsample, said first selective binding agent being present in an amountsufficient to reduce the concentration of free interfering ion in thefluid sample to insignificant levels, wherein the measured activity ofsaid enzyme is proportional to the concentration of the potassium ionsin the fluid sample.
 12. Process according to claim 11, wherein saidfluid sample is water.
 13. Process according to claim 11 wherein saidfluid sample is a body fluid.
 14. Process according to claim 13, whereinsaid body fluid is selected from the group consisting of blood, serum,plasma, urine, sweat, exudates or transudates.
 15. Process according toclaim 13, wherein the transferase is a microbial transferase. 16.Process according to claim 15, wherein the microbial transferase isbacterial pyruvate kynase.
 17. Process according to claim 16, whereinthe first selective binding agent is4,7,13,16,21-Pentaoxa-1,10-diazabicyclo 8.8.5.!-tricosan.
 18. Processaccording to claim 13 wherein the oxidoreductase is acetaldehydedehydrogenase or glycerol dehydrogenase.
 19. Process according to claim13, wherein the first selective binding agent is4,7,13,16,21-Pentaoxa-1,10-diazabicyclo 8.8.5.!-tricosan.
 20. Processaccording to claim 13, wherein said first selective binding agent isselected from the group consisting of cryptands, coronands, podands,crown ethers, spherands, hemispherands, calixarens, natural occurringionophores, cyclic peptides, complexones or chelating agents. 21.Process according to claim 13, wherein the first selective binding agentis 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo 8.8.5.!-tricosan.
 22. Processfor the determination of the concentration of potassium ions in a fluidsample, comprising measuring the activity of an enzyme whose activity isstimulated by potassium ions and is selected from the group consistingof a transferase or an oxidoreductase in the presence of a firstselective binding agent which binds to potassium ions in the fluidsample, said first selective binding agent being present in an amountsufficient to increase the ratio of the activity of the enzyme to theconcentration of free potassium ions in the fluid sample to within anoptimal range for measurement of the concentration of potassium ions inthe fluid sample, wherein the measured activity of said enzyme isproportional to the concentration of said potassium ion in the fluidsample.
 23. Process according to claim 22, wherein said fluid sample iswater.
 24. Process according to claim 22 wherein said fluid sample is abody fluid.
 25. Process according to claim 24, wherein said body fluidis selected from the group consisting of blood, serum, plasma, urine,sweat, exudates or transudates.
 26. Process according to claim 24,wherein the transferase is a microbial transferase.
 27. Processaccording to claim 26, wherein the microbial transferase is bacterialpyruvate kynase.
 28. Process according to claim 27, wherein the firstselective binding agent is 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo8.8.8!hexacosan.
 29. Process according to claim 24 wherein theoxidoreductase is acetaldehyde dehydrogenase or glycerol dehydrogenase.30. Process according to claim 29, wherein the first selective bindingagent is 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan. 31.Process according to claim 24, wherein said first selective bindingagent is selected from the group consisting of cryptands, coronantis,podands, crown ethers, spherands, hemispherands, caixarens, naturaloccurring ionophores, cyclic peptides, complexones or chelating agents.32. Process according to claim 24, wherein the first selective bindingagent is 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan. 33.Process for the determination of the concentration of potassium ions ina fluid sample, comprising measuring the activity of an enzymestimulated by an indicator ion, said enzyme selected from the groupconsisting of a transferase, a hydrolase, an oxidoreductase or a lyasein the presence of a selective binding agent and said indicator ion,wherein the selective binding agent forms a complex with the indicatorion and the indicator ion is displaced stoichiometrically from thecomplex by the potassium ion, whereby the displaced indicator ionstimulates the activity of the enzyme, wherein the activity of theenzyme is proportional to the concentration of potassium ions in thefluid sample.
 34. Process according to claim 1, further comprising thepresence of a first selective binding agent which binds to aninterfering ion in the fluid sample and is present in an amountsufficient to reduce the concentration of free interfering ion in thefluid sample to insignificant levels.
 35. Process according to claim 34,wherein said competitive inhibitor ion is a lithium ion and said firstselective binding agent is 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo8.8.5.!-tricosan.
 36. Process according to claim 34, further comprisinga second selective binding agent which binds to potassium ions in thefluid sample, said second selective binding agent and said competitiveinhibitor ion being present in amounts sufficient to increase the ratioof the activity of the enzyme to the concentration of free potassiumions in the fluid sample to within an optimal range for measurement ofthe concentration of potassiums ion in the fluid sample.
 37. Processaccording to claim 36, wherein said first selective binding agent is4,7,13,16,21-Pentaoxa-1,10-diazabicyclo 8.8.5.!-tricosan, said secondselective binding agent is 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo8.8.8!hexacosan, and said competitive inhibitor ion is a lithium ion.38. Process according to claim 1, further comprising the presence of afirst selective binding agent which binds to potassium ions in the fluidsample, said competitive inhibitor ion and said first selective bindingagent being present in amounts sufficient to increase the ratio of theactivity of the enzyme to the concentration of free potassium ions inthe fluid sample to within an optimal range for measurement of theconcentration of potassium ions in the fluid sample.
 39. Processaccording to claim 38, wherein said competitive inhibitor ion is alithium ion and said first selective binding agent is4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan.
 40. Processaccording to claim 11, further comprising the presence of a secondselective binding agent which binds to potassium ions in the fluidsample and is present in an amount sufficient to increase the ratio ofthe activity of the enzyme to the concentration of free potassium ionsin the fluid sample to within an optimal range for measurement of theconcentration of potassium ions in the fluid sample.
 41. Processaccording to claim 40, wherein said first selective binding agent is4,7,13,16,21-Pentaoxa-1,10-diazabicyclo 8.8.5.!-tricosara and saidsecond selective binding agent is4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan.
 42. Acomposition for the determination of potassium ions in a fluid samplecomprising;(a) an enzyme whose activity is stimulated by potassium ionsand is selected from the group consisting of a transferase or anoxidoreductase, and (b) a first selective binding agent that binds tointerfering ions and is present in a quantity sufficient to reduce theconcentration of free interfering ions in the fluid sample toinsignificant levels.
 43. Composition according to claim 42, wherein thetransferase is a microbial transferase.
 44. Composition according toclaim 43, wherein the microbial transferase is bacterial pyruvatekinase.
 45. Composition according to claim 44, wherein the firstselective binding agent is 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo8.8.5.!-tricosan.
 46. Composition according to claim 43 wherein theoxidoreductase is acetaldehyde dehydrogenase or glycerol dehydrogenase.47. Composition according to claim 46, wherein the first selectivebinding agent is 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo8.8.5.!-tricosan.
 48. Composition according to claim 42, wherein saidfirst selective binding agent is selected from the group consisting ofcryptands, coronands, podands, crown ethers, spherands, hemispherands,calixarens, natural occurring ionophores, cyclic peptides, complexonesor chelating agents.
 49. Composition according to claim 42, wherein thefirst selective binding agent is 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo8.8.5.!-tricosan.
 50. A composition for the determination of potassiumions in a fluid sample comprising;(a) an enzyme whose activity isstimulated by potassium ions and is selected from the group consistingof a transferase or an oxidoreductase, and (b) a first selective bindingagent that binds to potassium ions and is present in an amountsufficient to increase the ratio of the activity of the enzyme to theconcentration of free potassium ions in the fluid sample to within anoptimal range for measurement of the concentration of potassium ions inthe fluid sample.
 51. Composition according to claim 50, wherein thetransferase is a microbial transferase.
 52. Composition according toclaim 51, wherein the microbial transferase is bacterial pyruvatekynase.
 53. Composition according to claim 52, wherein the firstselective binding agent is 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo8.8.8!hexacosan.
 54. Composition according to claim 50 wherein theoxidoreductase is acetaldehyde dehydrogenase or glycerol dehydrogenase.55. Composition according to claim 54, wherein the first selectivebinding agent is 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo8.8.8!hexacosan.
 56. Composition according to claim 50, wherein saidfirst selective binding agent is selected from the group consisting ofcryptands, coronands, podands, crown ethers, spherands, hemispherands,calixarens, natural occurring ionophores, cyclic peptides, complexonesor chelating agents.
 57. Composition according to claim 50, wherein thefirst selective binding agent is4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan.
 58. Acomposition for the determination of the concentration of potassium ionsin a fluid sample comprising;(a) an enzyme whose activity is stimulatedby potassium ions and is selected from the group consisting of atransferase or an oxidoreductase, and (b) a competitive inhibitor ionwhich is present in an amount sufficient to increase the ratio of theactivity of the enzyme to the concentration of free potassium ions inthe fluid sample to within an optimal range for measurement of theconcentration of potassium ions in the fluid sample.
 59. Compositionaccording to claim 58, wherein the transferase is a microbialtransitrase.
 60. Composition according to claim 59, wherein themicrobial transferase is bacterial pyruvate kynase.
 61. Compositionaccording to claim 60, wherein the competitive inhibitor ion is alithium ion.
 62. Composition according to claim 58 wherein theoxidoreductase is acetaldehyde dehydrogenase or glycerol dehydrogenase.63. Composition according to claim 62, wherein said competitiveinhibitor ion is a lithium ion.
 64. Composition according to claim 58,wherein said competitive inhibitor ion is a lithium ion.
 65. Acomposition as claimed in claim 58 consisting essentially of:

    ______________________________________    Glycerol dehydrogenase                      50 U/l     to    100 U/l    Glycerol          0.3 mmol/l to    3 mmol/l    4,7,13,16,21-pentaoxa-1,10-                      0.06 mmol/l                                 to    30 mmol/l    diazabicyclo 8.8.5!tricosane    nicotinamide adenine                      0.1 mmol/l to    5.0 mmol/l    dinucleotide    buffer, pH 9      20 mmol/l  to    500 mmol/l    Serum albumin     0 g/l      to    5 g/l    glutamate dehydrogenase                      2,500 U/l  to    20,000 U/l    (assayed at 25° C.)    alpha-ketoglutarate (free acid)                      1 mmol/l   to    10 mmol/l.    ______________________________________


66. A composition as claimed in claim 58 consisting essentially of:

    ______________________________________    Acetaldehyde dehydrogenase                      50 U/l     to    10,000 U/l    Glycolaldehyde    0.3 mmol/l to    30 mmol/l    4,7,13,16,21-pentaoxa-1,10-                      0.06 mmol/l                                 to    30 mmol/l    diazabicyclo 8.8.5!tricosane    nicotinamide adenine                      0.05 mmol/l                                 to    2.0 mmol/l    dinucleotide    buffer, pH 7-8    50 mmol/l  to    500 mmol/l    Dithiothreitol    0.1 mmol/l to    2 mmol/l    Serum albumin     0 g/l      to    5 g/l    glutamate dehydrogenase                      2,500 U/l  to    20,000 U/l    (assayed at 25° C.)    alpha-ketoglutarate (free acid)                      1 mmol/l   to    10 mmol/l.    ______________________________________


67. A composition as claimed in claim 58 consisting essentially of:

    ______________________________________    Acetaldehyde dehydrogenase                      50 U/l     to    10,000 U/l    4-nitrophenyl acetate                      0.1 mmol/l to    2 mmol/l    4,7,13,16,21-pentaoxa-1,10-                      0.06 mmol/l                                 to    30 mmol/l    diazabicyclo 8.8.5!tricosane    reduced nicotinamide adenine                      0.001 mmol/l                                 to    0.1 mmol/l    dinucleotide    buffer, pH 7-8    50 mmol/l  to    500 mmol/l    Dithiothreitol    0.1 mmol/l to    2.0 mmol/l    Serum albumin     0 g/l      to    5 g/l    glutamate dehydrogenase                      2,500 U/l  to    20,000 U/l    (assayed at 25° C.)    alpha-ketoglutarate (free acid)                      1 mmol/l   to    10 mmol/l.    ______________________________________


68. Composition according to claim 58, further comprising the presenceof a first selective binding agent which binds to an interfering ion inthe fluid sample and is present in an amount sufficient to reduce theconcentration of free interfering ion in the fluid sample toinsignificant levels.
 69. Composition according to claim 68, whereinsaid competitive inhibitor ion is a lithium ion and said first selectivebinding agent is 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo8.8.5.!-tricosan.
 70. Composition according to claim 69, furthercomprising a second selective binding agent which binds to potassiumions in the fluid sample, said second selective binding agent and saidcompetitive inhibitor ion being present in amounts sufficient toincrease the ratio of the activity of the enzyme to the concentration offree potassium ions in the fluid sample to within an optimal range formeasurement of the concentration of potassium ions in the fluid sample.71. Composition according to claim 70, wherein said first selectivebinding agent is 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo8.8.5.!-tricosan, said second selective binding agent is4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan, and saidcompetitive inhibitor ion is a lithium ion.
 72. Composition according toclaim 58, further comprising the presence of a first selective bindingagent which binds to potassium ion in the fluid sample, said competitiveinhibitor ion and said first selective binding agent being present inamounts sufficient to increase the ratio of the activity of the enzymeto the concentration of free potassium ions in the fluid sample towithin an optimal range for measurement of the concentration ofpotassium ions in the fluid sample.
 73. Composition according to claim72, wherein said competitive inhibitor ion is a lithium ion and saidfirst selective binding agent is4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan.
 74. Acomposition as claimed in claim 68 consisting essentially of:

    ______________________________________    pyruvate kinase   50 U/l     to    10,000 U/l    (B. stearothermophilus)    phosphoenolpyruvate                      0.3 mmol/l to    30 mmol/l    (neutralized Tris salt)    4,7,13,16,21-pentaoxa-1,10-                      0.06 mmol/l                                 to    30 mmol/l    diazabicyclo 8.8.5!tricosane    reduced nicotinamide adenine                      0.01 mmol/l                                 to    0.8 mmol/l    dinucleotide    buffer, pH 7-8    50 mmol/l  to    500 mmol/l    Mn.sup.2+  or Mg.sup.2+                      1 mmol/l   to    10 mmol/l    LiCl              2 mmol/l   to    100 mmol/l    adenosine diphosphate (free acid)                      0.5 mmol/l to    10 mmol/l    lactate dehydrogenase (assayed                      5,000 U/l  to    100,000 U/l    at 25° C.)    Serum albumin     0 g/l      to    5 g/l    glutamate dehydrogenase                      2,500 U/l  to    20,000 U/l    (assayed at 25° C.)    alpha-ketoglutarate (free acid)                      1 mmol/l   to    10 mmol/l.    ______________________________________


75. Composition according to claim 42, further comprising the presenceof a second selective binding agent which binds to potassium ions in thefluid sample and is present in an amount sufficient to increase theratio of the activity of the enzyme to the concentration of freepotassium ions in the fluid sample to within an optimal range formeasurement of the concentration of potassium ions in the fluid sample.76. Composition according to claim 75, wherein said first selectivebinding agent is 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo8.8.5.!-tricosan and said second selective binding agent is4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo 8.8.8!hexacosan.
 77. Acomposition for the determination of the concentration of potassium ionsin a fluid sample, comprising an indicator ion, an enzyme stimulated bysaid indicator ion, said enzyme selected from the group consisting of atransferase, a hydrolase, an oxidoreductase or a lyase, and a selectivebinding agent that forms a complex with the indicator ion whereby theindicator ion is displaced stoichiometrically from the complex by saidpotassium ions in said fluid sample wherein the activity of the enzymeis proportional to the concentration of potassium ions in the fluidsample.